Figure 2.
Thermal stability of ϕ6 wt and E491Q RdRps. Thermal melting curves for wt and E491Q polymerases are shown, along with calculated melting temperature (Tm). Wild-type or E491Q were mixed with SYPRO Orange (Molecular Probes, Invitrogen) in starting buffer (50 mM Tris–HCl, 50 mM NaCl) alone and with the addition of either 5 mM EDTA, 10 mM MnCl2 or both 5 mM EDTA and 10 mM MnCl2. The Tm was calculated from the maximum value of the negative first derivative of fluorescence intensity versus temperature; this is approximately the midpoint of the unfolding transition (68). Any shift in Tm under different conditions to that of the starting buffer (i.e. in the presence of an additive), indicates a change in protein stability. An increase in Tm indicates a stabilization of the protein by an increase in structural order and a reduction in conformational flexibility, while a decrease in Tm indicates a destabilization (65).